The present investigation was undertaken to identify and characterize the tetracycline resistance determinant in 22 Gallibacterium anatis strains for which no determinant was identified using primers specific for tet(A, B, C, D, E, G, H, K, L, M, O). A recent study found tet(B) to be the most prevalent tetracycline resistance determinant in a larger collection of G. anatis field strains from Mexico and Denmark. However, in 41% of the tetracycline resistant strains no determinant could be assigned. Here we demonstrate that tet(31) is a common determinant in G. anatis originating from chickens from very different production systems and localities. In addition, tet(31) was identified in strains isolated over a 30-year period. This is the first report on tet(31) since its original identification in Aeromonas salmonicida.